WO2019213065A1 - Procédés de thérapie génique - Google Patents

Procédés de thérapie génique Download PDF

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WO2019213065A1
WO2019213065A1 PCT/US2019/029890 US2019029890W WO2019213065A1 WO 2019213065 A1 WO2019213065 A1 WO 2019213065A1 US 2019029890 W US2019029890 W US 2019029890W WO 2019213065 A1 WO2019213065 A1 WO 2019213065A1
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vector
cell
subject
nucleic acid
cyp
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PCT/US2019/029890
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Markus Grompe
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Oregon Health & Science University
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Priority to MX2020011470A priority Critical patent/MX2020011470A/es
Priority to EP19796748.2A priority patent/EP3787693A4/fr
Priority to US17/045,806 priority patent/US20210130832A1/en
Priority to CA3097914A priority patent/CA3097914A1/fr
Priority to JP2020561005A priority patent/JP2021522787A/ja
Priority to KR1020207034139A priority patent/KR20210005184A/ko
Priority to CN201980029600.1A priority patent/CN112135640A/zh
Priority to AU2019263055A priority patent/AU2019263055A1/en
Publication of WO2019213065A1 publication Critical patent/WO2019213065A1/fr

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Definitions

  • cytochrome p450 cytochrome p450
  • rAAV vectors are the dominant gene therapy platform.
  • rAAV vectors are designed to integrate therapeutic payloads into genomic loci of interest through homologous recombination.
  • precise gene editing by homologous recombination in vivo has poor efficiency, typically resulting in sub-therapeutic and impermanent gene editing.
  • rAAV vectors remain mostly episomal and are lost with cell division.
  • random integration of adeno-associated viruses has been associated with liver cancer in both animals and humans, indicating a risk of harm to not only hepatocytes, but any other tissues.
  • Figure 1 provides a general schematic showing the principle of achieving drug selection by inactivating the metabolism of an inert prodrug (protoxin) into a toxic metabolite.
  • the normal status of the cell is depicted on the left, where the prodrug (protoxin) is metabolized to a toxic metabolite.
  • the knockdown or knockout of CYP enzymes, cytochrome P450 reductase (CYPOR or POR), or beta-catenin (Ctnnbl) protects the cells and allows them to proliferate.
  • Figure 2 provides a graph of blood alanine aminotransferase (ALT) levels, an indicator of liver damage, over time in mice injected with pX330-Cypor and treated with acetaminophen.
  • ALT blood alanine aminotransferase
  • Figure 3 provides CYPOR immunohistochemistry images of liver samples from mice transduced with pX330-Cypor that were either untreated (top image) or treated with acetaminophen (bottom image). Dashed line outlines CYPOR null nodules.
  • Figure 1 provides a general schematic showing the principle of achieving drug selection by inactivating the metabolism of an inert prodrug into a toxic metabolite.
  • the normal status of the cell is depicted on the left.
  • the metabolizing enzyme is expressed in normal cells, resulting in toxicity after metabolic conversion. If the gene(s) responsible for the metabolic activity are either knocked down (e.g., siRNA or shRNA) or knocked out (e.g., targeted nuclease (e.g., CRISPR)), the toxic metabolite is not produced after administration of the toxic precursor. Knockdown or knockout cells are protected and can proliferate, whereas enzyme positive cells die.
  • knocked down e.g., siRNA or shRNA
  • knocked out e.g., targeted nuclease (e.g., CRISPR)
  • CYP7A1 CYP7B
  • CYP8B e.g, CYP8B1
  • CYP11A e.g,
  • RNA-guided CRISPR/Cas9 system involves expressing Cas9 along with a guide RNA molecule (gRNA). Guidelines and computer-assisted methods for generating gRNAs are available (see, e.g, CRISPR Design Tool (crispr.mit.edu); Hsu et al. (2013) Nat. Biotechnok 31 :827-832;
  • PAM protospacer-adjacent motif
  • the nucleic acid molecules of the instant invention may be contained within a vector (e.g., plasmid, transposon, viral vector (e.g., adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses), etc.).
  • a vector e.g., plasmid, transposon, viral vector (e.g., adeno-associated viruses (AAVs), adenoviruses, retroviruses, and lentiviruses), etc.
  • AAVs adeno-associated viruses
  • retroviruses retroviruses
  • lentiviruses lentiviruses
  • the desired nucleic acid sequences may be expressed from appropriate promoters within the vector including a strong promoter, a constitutive promoter, tissue or cell specific promoter (e.g., hepatocyte specific promoter), and/or a regulated promoter.
  • promoters are well known in the art and include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and RNA polymerase III promoters (e.g., U6 and Hl; see, e.g., Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09).
  • the desired nucleic acid sequences are expressed from an endogenous promoter (e.g., a promoter within the cell).
  • the parental compound is inert and non-toxic (e.g., a prodrug), but its metabolite(s) generated by Cyp mediated enzymatic conversion are toxic, especially hepatotoxic.
  • acetaminophen is metabolized to the hepatotoxic compound N-acetyl-p-benzoquinone imine (NAPQI) by three POR dependent CYP enzymes (CYP1A2, CYP2E1, and CYP3A4).
  • Cyp dependent toxins are used for in vivo selection of gene modified hepatocytes or other modified cells, in which the Cyp activity responsible for producing the toxic metabolite has been inactivated. Examples of CYP dependent toxins are provided in Table 1.
  • the CYP dependent toxin may include acetaminophen (APAP) as the prodrug that is converted to the active drug by any of: CYP2E1, CYP1 A2, CYPA4, or CYP2D6.
  • the CYP dependent toxin may include retrosine as the prodrug that is converted to the active drug by CYP3 A4.
  • the CYP dependent toxin may include tangretin.
  • the CYP dependent toxin may include cyclopohsphamide as the prodrug that is converted to the active drug phosphoramide mustard by hydroxylation facilitated by any of: CYP2B6, CYP2C9, or CYP3 A4.
  • the CYP dependent toxin may include ifosfamide as the prodrug that is converted to the active drug ifosfamide mustard by hydroxylation.
  • the CYP dependent toxin may include trofosfamide as the prodrug that is converted to the active drug trofosfamide mustard by hydroxylation.
  • the CYP dependent toxin may include pradefovir as the prodrug that is converted to the active drug PMEA-triphosphate by hydroxylation.
  • the CYP dependent toxin may include MB07133 as the prodrug that is converted to the active drug araC -triphosphate by hydroxyl ati on.
  • the CYP dependent toxin may include sibrafiban as the prodrug that is converted to the active drug Ro 48-3888 mustard by reduction.
  • the CYP dependent toxin may include ximelagatran as the prodrug that is converted to the active drug melagartan by reduction.
  • the CYP dependent toxin may include guanoxabenz as the prodrug that is converted to the active drug guanabenz by reduction.
  • the CYP dependent toxin may include AQ4N as the prodrug that is converted to the active drug AQ4 by reduction.
  • the CYP dependent toxin may include tamoxifen as the prodrug that is converted to the active drug 4-hydroxy-tamoxifen by hydroxylation facilitated by CYP3 A4. In another aspect, the CYP dependent toxin may include tamoxifen as the prodrug that is converted to the active drug N-Desmethyl-tamoxifen by N-demethylation. In another aspect, the CYP dependent toxin may include tamoxifen as the prodrug that is converted to the active drug endoxifen by hydroxylation or N-demethylation. In another aspect, the CYP dependent toxin may include ketoconazole as a prodrug that is converted to an active drug by CYP3 A4.
  • a) administering a donor molecule comprising:
  • a gRNA e.g., a scgRNA
  • the gRNA sequence combines with a cas9 nuclease, resulting in an active, site-nuclease designed to cut, knock down, knock out, and/or otherwise disrupt a CYP enzyme(s)
  • Cypor gene or the Ctnnb 1 gene
  • a polymerase II promoter e.g., a cell and/or tissue specific promoter
  • a cell and/or tissue specific Homology Arm sequence designed to facilitate, promote, or otherwise enable homologous integration into a desired recipient cell locus
  • cas9 nuclease e.g., protein or nucleic acid encoding Cas9
  • cas9 nuclease e.g., protein or nucleic acid encoding Cas9
  • the cas9 nuclease combines with a gRNA resulting in an active, site- nuclease designed to cut, knock down, knock out, and/or otherwise disrupt the a CYP enzyme(s), the Cypor gene, or the Ctnnb 1 gene
  • a gRNA sequence into a DNA donor molecule wherein one or more ribozyme sequences are embedded upstream from the gRNA sequence (“left ribozyme”) and one or more ribozyme sequences are embedded downstream from the gRNA sequence (“right ribozyme”), an unspliced Pol2 RNA transcript containing gRNA sequence is properly processed by the combined cleaving by the left ribozyme and the right ribozyme.
  • the non-random, simultaneous co-expression of functional gRNA and transgenes targeted to specific cell and/or tissue types is made possible by a scgRNA molecule.
  • gRNA sequence combines with a cas9 nuclease, resulting in an active, site-nuclease designed to cut, knock down, knock out, and/or otherwise disrupt a CYP enzyme(s) or the Cypor Gene; iii) promoter;
  • prodrug e.g., a CYP dependent toxin
  • methods of expressing a gRNA comprise expressing a self-cleaving guide RNA (scgRNA) under the control of a pol2 promoter (e.g., a tissue or cell specific pol2 promoter).
  • a self-cleaving gRNA comprises ribozymes at the 5’ and 3’ ends of a single guide RNA.
  • the self-cleaving ribozymes release the gRNA within the cell to act with Cas9.
  • nucleic acid or a“nucleic acid molecule” as used herein refers to any
  • nucleic acid molecules DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • the term“isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an“isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • antisense molecules are typically between about 15 and about 50 nucleotides in length, more particularly between about 15 and about 30 nucleotides, and often span the translational start site of mRNA molecules.
  • Antisense constructs may also be generated which contain the entire sequence of the target nucleic acid molecule in reverse orientation.
  • Antisense oligonucleotides targeted to any known nucleotide sequence can be prepared by oligonucleotide synthesis according to standard methods. Antisense oligonucleotides may be modified as described above to comprise nuclease resistant modifications.
  • embedded means the inclusion of certain desired oligonucleotide sequence within a larger synthetic oligonucleotide, forming a single molecule, wherein said molecule may or may not have additional activity or purpose beyond that of the embedded sequence.
  • “homology arms” means target sequence with homology to a desired gene locus designed to promote, facilitate, and enable homologous integration of a donor molecule into a recipient cell genome.
  • knockout means a technique by which an organism’s gene is made inoperative.
  • knockdown means a technique by which the expression of an organism’s gene is reduced.
  • Linker refers to a chemical moiety comprising a chain of atoms that covalently attach at least two compounds.
  • the linker can be linked to any synthetically feasible position of the compounds, but preferably in such a manner as to avoid blocking the compounds desired activity.
  • Linkers are generally known in the art.
  • the linker may contain from 1 to about 50 atoms, from 1 to about 10 atoms, or from about 1 to about 5 atoms.
  • non-integrating vector means a vector that delivers donor molecules, transgenes, or other exogenous DNA or RNA wherein said donor molecules remain episomal and/or are not integrated into a recipient cell chromosome or genome.
  • downstream or means a genetic locus or sequence position that is located closer the 5' -terminus in relation to the reference locus, wherein the oligonucleotide sequence begins with the 5’-terminus and runs“5’ to 3’” until ending at the 3’-terminus.
  • Plasmid construction A CRISPR recognition site was identified within exon 1 of the mouse Cypor gene using an online design tool (available at
  • pX330 is a human codon-optimized SpCas9 and chimeric guide RNA expression plasmid (Cong, et al. Science (2013) 339(6l2l):819-23). Briefly, the plasmid comprises the gRNA under control of the U6 promoter and SpCas9 under the control of the CBh promoter (a hybrid chicken beta actin promoter).
  • the oligonucleotide sequences were 5’-CACCGTCGTGGGGGTCCTGACCTAC-3’ (SEQ ID NO: 519) and 5’- AAACGTAGGTCAGGACCCCCACGAC-3’ (SEQ ID NO: 520).
  • the resulting plasmid (pX330-Cypor) was validated to cut the targeted sequence using the Guide- ItTM Mutation Detection kit (Takara Bio USA, Inc., Mountain View, CA).
  • ALT levels were measured 6-20 hours following APAP injection from 10 m ⁇ of blood drawn from a saphenous vein puncture using the ALT Reagent Kit (available at BKKits.com, BQ 004A-CR). Mice were sacrificed two weeks after the final APAP injection and the livers were harvested.
  • liver damage as measured by blood ALT levels decreased over time for the mice injected with pX330-Cypor.
  • the treated mice were APAP resistant after -10 weeks.
  • CYPOR immunohistochemistry results are provided in Figure 3.
  • pX330-Cypor treated mice that received APAP injections distribution with large CYPOR-negative nodules are present, thereby indicating clonal expansion of Cypor-null hepatocytes (bottom image; dashed line outlines Cypor null nodules).
  • the large CYPOR-negative nodules are not present in mice not treated with APAP (top image).
  • Figure 4 shows the number of Cypor indels in mice injected with (from left to right) a control plasmid (pX330), pX330-Cypor without APAP selection, pX330- Cypor with APAP injections, and pX330-Cypor with an APAP diet.
  • Indel frequency in APAP treated mice was significantly higher (p ⁇ 0.01) in mice receiving APAP, either by injection or diet, thereby demonstrating selection of hepatocytes with the Cypor knockout.
  • the Sleeping Beauty transposon system was used to test CYPOR
  • mice were injected with two separate plasmids.
  • One plasmid contained sleeping beauty transposase under the control of the CMV promoter.
  • the second plasmid contained transposon cassette comprising a GFP expression cassette and U6 driven Cypor shRNA. After treatment, injected mice were exposed to 3 months of APAP selection.
  • Figure 6 provides CYPOR immunohistochemistry and GFP fluorescence on livers from mice in the experiments treated with the Sleeping Beauty transposon system and APAP.
  • the top panels show results from a negative control (irrelevant shRNA). Rare GFP+ cells can be seen, but no nodules have formed.
  • the bottom panels show images from a APAP treated mice that were injected with the GFP- Cypor shRNA transposon. Large confluent areas of GFP expression are seen in Cypor negative regions. This provides clear evidence for the expansion of transposon positive hepatocytes.
  • Self-cleaving guide RNAs were also tested.
  • Self-cleaving guide RNAs comprise ribozymes on either side of the guide RNA so that the guide RNA may be driven from a pol II promoter.
  • a hammerhead (HH) ribozyme may be at the 5’ of the guide RNA and a Hepatitis delta virus (HDV) ribozyme may be at the 3’ end of the guide RNA.
  • Figure 7 provides a schematic of the self-cleaving guide RNA.
  • a scgRNA comprising a guide RNA flanked by a hammerhead type ribozyme and a hepatitis delta virus type ribozyme was used.
  • the targeting vector utilized for these experiments contains human Factor IX as a biomarker and the scgRNA between arms of homology to the murine albumin locus.
  • the albumin gene was selected as an example of a cell-type specific gene locus.
  • Vectors were designed to contain scgRNA targeting either exon 3 of 4-OH phenylpyruvate di oxygenase (Hpd), exon 4 of Hpd or exon 4 of homogentisic acid di oxygenase (Hgd).
  • the vector comprised, in order: left arm of homology (albumin) - p2A (2A peptide) - human Factor IX gene - region of homology
  • mice were cycled on 2-(2-nitro-4- trifluoromethylbenzoyl)-l,3- cyclohexanedione (NTBC) to allow selection of hepatocytes that have the correct integration of the targeting vector and hence express gRNAs capable of disrupting selectable genes.
  • NTBC 2-(2-nitro-4- trifluoromethylbenzoyl)-l,3- cyclohexanedione
  • loss of Hpd or Hgd confers a selective growth advantage in hepatocytes.
  • Levels of circulating human Factor IX were assessed at 5, 9, 14 and 18 weeks of age. At 5 and 9 weeks, all mice displayed low levels of human Factor IX.
  • mice that had received the scgRNA vector with a gRNA against exon 3 of Hpd began to show increased levels of hF9.
  • mice that received the scgRNA vector with a gRNA targeting exon 4 of Hpd or Hgd showed high levels of hF9 by 18 weeks of age.
  • the albumin locus was targeted with a GeneRideTM vector incorporating self-cleaving guide RNA against Cypor. In initial experiments, the transgene luciferase was utilized.
  • the transgene and the self-cleaving guide RNA are expressed.

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CN112047990B (zh) * 2020-05-11 2021-06-04 西安新通药物研究股份有限公司 阿糖胞苷前药mb07133晶型及其应用
WO2023069423A3 (fr) * 2021-10-18 2023-09-28 Logicbio Therapeutics, Inc. Thérapie génique pour traiter la ht1

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